Method of cloning bovine embryos

Abstract

An improved method of cloning mammalian embryos by transferring a nucleus from a donor mammalian embryo to an enucleated recipient oocyte and culturing the nuclear transferred bovine embryo in vitro is described. The improvement includes positioning the donor embryo nucleus adjacent to the enucleated recipient oocyte, and maintaining the nucleus and oocyte in a maintenance medium for a time sufficient to mature the cytoplasm of the enucleated recipient oocyte prior to fusing the donor nucleus to the recipient oocyte.

What is claimed is: 
     
       1. A method for producing cloned bovine embryos comprising the following steps: a. removing the chromosomal material from a metaphase II stage bovine oocyte to form an enucleated recipent oocyte;   b. maintaining the enucleated recipient oocyte in CR-1+1 mM L-glutamine maintenance medium wherein the maintenance period is approximately 6 to 18 hours;   c. placing a membrane bound nucleus from a donor 2-64 cell bovine embryo adjacent to the plasma membrane of the enucleated recipient oocyte and;   d. inducing cell fusion between the membranes of the donor membrane-bound nucleus and the enucleated recipient oocyte to form an embryonic single cell with a nucleus from the donor, wherein the membranes are either electrically fused or fused with polyethylene glycol.   
     
     
       2. The method of claim 1 wherein the CR-1+1 mM L-glutamine maintenance media further comprises 5 mM hemicalcium L-lactage and 0.4 mM pyruvate. 
     
     
       3. The method of claim 1 further comprising, before step. d., orienting the donor membrane-bound nucleus and the enucleated recipient oocyte so that the plane of contact of their membranes is perpendicular to the direction of electric current flow.